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Protein phosphorylation is one of the most widespread post-translational modifications in biology 1 , 2 . With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes 3 , 4 . For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible 3 . Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways. Analysis of the kinase activity of 300 protein Ser/Thr kinases reveals that the substrate specificity of the kinome is substantially more diverse than expected and is driven extensively by negative selectivity

L-serine is a nonessential amino acid in eukaryotic cells, used for protein synthesis and in producing phosphoglycerides, glycerides, sphingolipids, phosphatidylserine, and methylenetetrahydrofolate. Moreover, L-serine is the precursor of two relevant coagonists of NMDA receptors: glycine (through the enzyme serine hydroxymethyltransferase), which preferentially acts on extrasynaptic receptors and D-serine (through the enzyme serine racemase), dominant at synaptic receptors. The cytosolic “phosphorylated pathway” regulates de novo biosynthesis of L-serine, employing 3-phosphoglycerate generated by glycolysis and the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase (the latter representing the irreversible step). In the human brain, L-serine is primarily found in glial cells and is supplied to neurons for D-serine synthesis. Serine-deficient patients show severe neurological symptoms, including congenital microcephaly, psychomotor retardation, and intractable seizures, thus highlighting the relevance of de novo production of this amino acid in brain development and morphogenesis. Indeed, the phosphorylated pathway is strictly linked to cancer. Moreover, L-serine has been suggested as a ready-to-use treatment, as also recently proposed for Alzheimer’s disease. Here, we present our current state of knowledge concerning the three mammalian enzymes of the phosphorylated pathway and known mutations related to pathological conditions: although the structure of these enzymes has been solved, how enzyme activity is regulated remains largely unknown. We believe that an in-depth investigation of these enzymes is crucial to identify the molecular mechanisms involved in modulating concentrations of the serine enantiomers and for studying the interplay between glial and neuronal cells and also to determine the most suitable therapeutic approach for various diseases.

Ubiquitin, known for its role in post-translational modification of other proteins, undergoes post-translational modification itself; after a decrease in mitochondrial membrane potential, the kinase enzyme PINK1 phosphorylates ubiquitin at Ser 65, and the phosphorylated ubiquitin then interacts with ubiquitin ligase (E3) enzyme parkin, which is also phosphorylated by PINK1, and this process is sufficient for full activation of parkin enzymatic activity. Phosphorylated ubiquitin is a parkin activator The small protein ubiquitin, familiar for its role in post-translational modification of other proteins by binding to them and regulating their activity or stability, is shown here to be the substrate of the kinase PINK1, which together with the ubiquitin ligase parkin is a causal gene for hereditary recessive Parkinsonism. Noriyuki Matsuda and colleagues show that following a decrease in mitochondrial membrane potential, PINK1 phosphorylates ubiquitin at serine residue 65; the phosphorylated ubiquitin then interacts with parkin, which is also phosphorylated by PINK1. This interaction allows full activation of parkin enzymatic activity, which involves tagging mitochondrial substrates with ubiquitin. PINK1 (PTEN induced putative kinase 1) and PARKIN (also known as PARK2 ) have been identified as the causal genes responsible for hereditary recessive early-onset Parkinsonism 1 , 2 . PINK1 is a Ser/Thr kinase that specifically accumulates on depolarized mitochondria, whereas parkin is an E3 ubiquitin ligase that catalyses ubiquitin transfer to mitochondrial substrates 3 , 4 , 5 . PINK1 acts as an upstream factor for parkin 6 , 7 and is essential both for the activation of latent E3 parkin activity 8 and for recruiting parkin onto depolarized mitochondria 8 , 9 , 10 , 11 , 12 . Recently, mechanistic insights into mitochondrial quality control mediated by PINK1 and parkin have been revealed 3 , 4 , 5 , and PINK1-dependent phosphorylation of parkin has been reported 13 , 14 , 15 . However, the requirement of PINK1 for parkin activation was not bypassed by phosphomimetic parkin mutation 15 , and how PINK1 accelerates the E3 activity of parkin on damaged mitochondria is still obscure. Here we report that ubiquitin is the genuine substrate of PINK1. PINK1 phosphorylated ubiquitin at Ser 65 both in vitro and in cells, and a Ser 65 phosphopeptide derived from endogenous ubiquitin was only detected in cells in the presence of PINK1 and following a decrease in mitochondrial membrane potential. Unexpectedly, phosphomimetic ubiquitin bypassed PINK1-dependent activation of a phosphomimetic parkin mutant in cells. Furthermore, phosphomimetic ubiquitin accelerates discharge of the thioester conjugate formed by UBCH7 (also known as UBE2L3) and ubiquitin (UBCH7∼ubiquitin) in the presence of parkin in vitro , indicating that it acts allosterically. The phosphorylation-dependent interaction between ubiquitin and parkin suggests that phosphorylated ubiquitin unlocks autoinhibition of the catalytic cysteine. Our results show that PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity. These findings demonstrate that phosphorylated ubiquitin is a parkin activator.

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3’end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m 6 A, the most abundant RNA modification. RBM15 positively regulates m 6 A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes. Here the authors establish the SPOC domain as a universal reader of the RNA Pol II CTD code and a versatile reader of phosphoserine marks found in co- and post-transcriptional regulators such as m6A writer and reader proteins.

Ovarian cancer (OC) is one of the leading causes for cancer-related deaths among women. MicroRNAs (miRs) have been proved to be vital to the development and progression of OC. Hence, the study aims to evaluate the ability of miR-195-5p affecting cisplatin (DDP) resistance and angiogenesis in OC and the underlying mechanism. MiRs that could target phosphoserine aminotransferase 1 (PSAT1), a differentially expressed gene in OC, were predicted by miRNA-mRNA prediction websites. The expression patterns of miR-195-5p in the OC tissues and cells were determined using RNA quantification assay. The role of miR-195-5p in OC was evaluated by determining DDP resistance, apoptosis and angiogenesis of OC cells after up-regulating or down-regulating miR-195-5p or PSAT1, or blocking the glycogen synthase kinase-3β (GSK3β)/β-catenin signaling pathway. Animal experiment was conducted to explore the effect of miR-195-5p on resistance to DDP and angiogenesis. MiR-195-5p directly targeted PSAT1 and down-regulated its expression. The expression of miR-195-5p was lower while that of PSAT1 was higher in OC tissues than in adjacent normal tissues. When miR-195-5p was over-expressed or PSAT1 was silenced, the expression of HIF-1α, VEGF, PSAT1, β-catenin as well as the extent of GSK3β phosphorylation was reduced, the angiogenesis and resistance to DDP was diminished and apoptosis was promoted both in vitro and in vivo. The inhibition of GSK3β/β-catenin signaling pathway was involved in the regulation process. Over-expression of miR-195-5p reduced angiogenesis and DDP resistance in OC, which provides a potential therapeutic target for the treatment of OC.

Intestinal bacteria can convert indole to a range of compounds; however, the precise enzymatic processes facilitating this conversion have not been fully elucidated. Certain Bifidobacterium strains convert exogenous indole to indole-3-lactic acid (ILA) via tryptophan synthase beta chain and aromatic lactate dehydrogenase. Moreover, the metabolism of indole is enhanced when the strain is combined with non-digestible oligosaccharides, forming synbiotics. However, the mechanism by which synbiotics enhance indole metabolism remains unclear. Here, the conversion of indole to ILA was investigated in the context of synbiotic-mediated enhancement. The combination of Bifidobacterium bifidum YIT 10347 and galactooligosaccharides (non-digestible oligosaccharides) in human fecal suspension medium synergistically increased this conversion. Screening Tn5 transposon-mutated B. bifidum YIT 10347 clones revealed that phosphoserine phosphatase B (PSP), encoded by serB , and tryptophan synthase alpha chain (TrpA), encoded by trpA , play crucial roles in indole metabolism. Mutant strains lacking either PSP or TrpA showed a significant reduction in ILA production, confirming the roles of PSP and TrpA in this pathway. Additionally, serine supplementation restored ILA production in PSP-deficient strains, further supporting the role of serine biosynthesis in indole metabolism. These results suggest that PSP and TrpA play a vital role in the metabolism of indole to ILA. This study provides novel insights into microbial indole metabolism and suggests potential applications of synbiotics in improving health. Key points • PSP and TrpA are essential for indole metabolism •  Bifidobacterium bifidum YIT 10347 and GOS synergistically metabolize indole to ILA •  This pathway offers potential therapeutic benefits for gut and kidney health Graphical Abstract

Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (Pf) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the Pf of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121.

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either TSC1 or TSC2. Cytokine profiling of TSC2-deficient LAM patient–derived cells revealed striking up-regulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed up-regulation of IL-6 transcription and secretion compared to wild-type cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-13C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wild-type cells. Treatment with anti–IL-6 (αIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in Tsc2 +/− mice while reducing PSAT1 expression. These data reveal a mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.

BackgroundAurora kinase B (AURKB), a key mitotic regulator, is frequently overexpressed in multiple malignancies including colorectal cancer (CRC) and correlates with poor prognosis. However, clinical trials have shown limited efficacy of AURKB inhibitors, indicating unrecognized oncogenic mechanisms that warrant further exploration.MethodsThe expression pattern of AURKB and its prognostic significance were explored in CRC tissues from our center and GEO cohort. The oncogenic roles of AURKB were demonstrated by proliferation and apoptosis assays. RNA sequencing (RNA-seq) and gene set enrichment analysis (GSEA) were performed to determine the downstream targets of AURKB. Mass spectrometry, Co-IP, RIP-qPCR, and mRNA stability assays were conducted to delineate the interplay and potential mechanisms involving AURKB, heterogeneous nuclear ribonucleoprotein M (HNRNPM), and phosphoserine aminotransferase 1 (PSAT1). The role of the AURKB/PSAT1 axis in CRC was further validated through in vivo rescue experiments.ResultsAURKB is highly expressed in CRC tissues and significantly correlates with unfavorable prognosis (IDDF2025-ABS-0178 figure 1. The expression pattern and prognostic significance of AURKB in CRC). Functional studies demonstrate that AURKB knockdown suppresses CRC proliferation and induces apoptosis (IDDF2025-ABS-0178 figure 2. AURKB depletion inhibits CRC proliferation and induces apoptosis). RNA-seq and GSEA reveal that AURKB regulates serine/glycine metabolism. Subsequent experiments show that AURKB depletion reduces PSAT1 expression and serine biosynthesis, an effect not observed with the kinase inhibitor AZD2811, suggesting a kinase-independent mechanism (IDDF2025-ABS-0178 figure 3. AURKB knockdown attenuates PSAT1 expression and serine biosynthesis). Mechanistically, AURKB interacts with HNRNPM and disrupts its binding to PSAT1 mRNA, thereby attenuating HNRNPM-mediated mRNA degradation and enhancing PSAT1 protein levels (IDDF2025-ABS-0178 figure 4. AURKB inhibits HNRNPM-mediated PSAT1 mRNA degradation). In vivo, rescue experiments confirm that PSAT1 overexpression reverses the effects of AURKB depletion on CRC progression (IDDF2025-ABS-0178 figure 5. PSAT1 rescues the proliferation suppression induced by AURKB depletion).Abstract IDDF2025-ABS-0178 Figure 1The expression pattern and prognostic significance of AURKB in CRC[Figure omitted. See PDF]Abstract IDDF2025-ABS-0178 Figure 2AURKB depletion inhibits CRC proliferation and induces apoptosis[Figure omitted. See PDF]Abstract IDDF2025-ABS-0178 Figure 3AURKB knockdown attenuates PSAT1 expression and serine biosynthesis[Figure omitted. See PDF]Abstract IDDF2025-ABS-0178 Figure 4AURKB inhibits HNRNPM-mediated PSAT1 mRNA degradation[Figure omitted. See PDF]Abstract IDDF2025-ABS-0178 Figure 5PSAT1 rescues the proliferation suppression induced by AURKB depletion[Figure omitted. See PDF]ConclusionsThis study reveals a kinase-independent role for AURKB in CRC through its interaction with RNA-binding protein, redefining its therapeutic potential beyond kinase inhibition. These findings highlight the need for alternative targeting strategies, such as PROTAC-based AURKB degraders, or pharmacological inhibition of the AURKB/PSAT1 axis, to fully harness its role in CRC treatment.

Alterations in cellular ubiquitin （Ub） homeostasis, known as Ub stress, feature and affect cellular responses in multiple conditions, yet the underlying mechanisms are incompletely understood. Here we report that autophagy receptor p62/sequestosome-1 interacts with E2 Ub conjugating enzymes, UBE2D2 and UBE2D3. Endogenous p62 undergoes E2-dependent ubiquitylation during upregulation of Ub homeostasis, a condition termed as Ub~ stress, that is intrinsic to Ub overexpression, heat shock or prolonged proteasomal inhibition by bortezomib, a chemothera- peutic drug. Ubiquitylation of p62 disrupts dimerization of the UBA domain of p62, liberating its ability to recognize polyubiquitylated cargoes for selective autophagy. We further demonstrate that this mechanism might be critical for autophagy activation upon Ub stress conditions. Delineation of the mechanism and regulatory roles of p62 in sensing Ub stress and controlling selective autophagy could help to understand and modulate cellular responses to a variety of endogenous and environmental challenges, potentially opening a new avenue for the development of therapeutic strategies against autophagy-related maladies.